1. Field of the Invention
The present application relates to process of making clofarabine, which is an active pharmaceutical ingredient.
2. Description of the Related Art
Clofarabine is the active pharmaceutical ingredient (API; drug substance) in the anticancer drug product Clolar®, which was originally developed by Ilex Oncology. Clolar® was approved for sale by the FDA in 2004 for treating children with refractory or relapsed acute lymphoblastic leukemia. Clofarabine is a fluoro-deoxy arabinonucleoside, which is a synthetic analogue of adenosine.
Wright et al.1 reported the synthesis of 9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-adenine (1), which is simply the 2-dechloro analogue of clofarabine, by acid catalysed (p-TsOH) fusion (coupling) of 1,3-di-O-acetyl-5-β-benzyl-2-deoxy-2-fluoro-D-arabinofuranside (2), which was prepared from methyl 2,3-anhydro-α-D-ribofuranoside (3),2 with 2,6-dichloropurine (4) (notice that this is the free purine, i.e., it is not silylated or deprotonated) to give a 30% isolated yield of β-N9 and 29% α-N9 by short column chromatography, followed by amination and reduction reaction (Scheme 2). An important aspect and disadvantage to this process is the fact that a 1:1 mixture of α- and β-anomers are formed (only the β-anomer is desired) and the follow-on effect from this is that column chromatography is needed to separate the isomers. This makes such a process not amenable to scale-up due to the cost of large scale-up chromatography. Another aspect of note is that the choice of benzyl protection of the C5′-OH meant that the process could not be used to synthesize clofarabine because hydrogenolysis of benzyl group leads to simultaneous removal of the requisite C2-chlorine atom on the adenine ring. That is, benzyl groups and the chlorine atom of the adenine ring are not orthogonal. Thus, the starting material 2 could not be used to synthesize clofarabine.
Watanable et al.3 disclose a synthesis approach to 2′-deoxy-2′-fluoro-arabinofuranosyl purine nucleosides under solution conditions without a catalyst utilizing 2 as a starting material, but clofarabine was not accessible using this route.

Perhaps in an effort to solve this problem, Reichman et al.4 synthesized 3-O-acetyl-5-O-benzoyl-2-deoxy-2-fluoro-β-D-arabinofuranosyl bromide (5a), by a long multi-step procedure (Scheme 3), which instead exchanged the troublesome benzyl group for a benzoyl group.

Montgomery et al.5 later successfully utilized this differently protected 1-α-bromo carbohydrate 5a in the synthesis of protected dichloropurine-based nucleoside 6 through the coupling of free 2,6-dichloropurine (4) in DCE in at 100° C. over a 16-hour period in the presence of molecular sieves (Scheme 4). Although the desired β-anomer of 6 was the major product other nucleosides including the β-anomer of 6 were also formed, once again showing pre-fluorination of the carbohydrate ring leads to an inherently inefficient coupling step. Pure protected nucleoside 6 was only obtained after the purification by column chromatography in 32% yield from intermediate 5a, and therefore in lower yield based on the true carbohydrate starting material. The authors also tested the 1-O—Ac-desbromo analogue 5b in the coupling with 4 but this did not provide an acceptable yield of the desired β-anomer of 6.

At the same time that Montgomery utilized 1-α-bromo sugar 5a as a starting material in the synthesis of dichloropurine nucleoside 6 (above), Howell et al.6 instead utilized the close analogue 2-deoxy-2-fluoro-3,5-di-O-benzoyl-α-D-arabino-furanosyl bromide (8) (Scheme 5). These two compounds, viz. 5a and 8, differ only by the acyl protecting group positioned at C3-0. Howell et al.'s bromosugar 8 was prepared in 4 synthetic steps in 33-43% overall yield (43% if a recycle is used) from 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose (9; which is the same starting material as used in our synthesis of clofarabine) using rearrangement, sulfonylation, fluorination, and finally bromination steps (Scheme 5). This bromosugar 8 has since been the key starting material in most clofarabine syntheses that we are aware of, but can also be used in the synthesis of other nucleosides such as a series of (2′-fluoro-2′-deoxy-β-D-ribofuranosyl)-uracils 10a-d.7

Montgomery was the first to disclose a method for the synthesis of clofarabine (7).8 The method comprised using his already disclosed7 uncatalysed coupling of 1-α-bromo sugar 5a with 2,6-dichloropurine (4), followed by a dual amination and deprotection step (Scheme 6). The coupling reaction of 4 and 5a at reflux in DCE gave an anomeric mixture of N9 isomers from which the desired N9 β-anomer isomer of intermediate 6 was obtained in 32% yield after column chromatography. Amination and deprotection of the desired N9 β-anomer of 6 gave clofarabine (7). Amination by itself provided a mono-benzoylated clofarabine intermediate (i.e., amination only deprotected the C3′-OAc group and substituted the C6-Cl group) which had to be further deprotected by the addition of LiOH to give 7. Three recrystallization from water gave pure 7 in 42% yield. The overall yield of clofarabine was only 13% based on 5a, and therefore in lower yield based on the carbohydrate starting material that was used to make 5a itself.
Not only was the longwinded purification of 6 and 7 not fit for commercial production on scale-up, but the synthesis of the starting material carbohydrate 5a6 was complicated. Moreover, the instability of 5a was also a disadvantage for scale-up and the coupling reaction had to be run under very dry (i.e., low levels of water) conditions, since otherwise 5a partially decomposed during the reaction.

Much more recently, Montgomery reported an improved method that involved the coupling of the sodium salt 17, or other salts (such as formed using DBU), of 2-chloro-6-substituted purine (due to the relatively high acidity of the N9 hydrogen) with the now preferred 2-deoxy-2-fluoro-3,5-di-O-benzoyl-α-D-arabinofuranosyl bromide (8) to give an anomeric mixture of intermediate 11 (Scheme 7).9 One key and obvious difference between this and the older procedure was the use of an anionic salt of the purine, which would function to render it more reactive. Indeed, the coupling reaction could be conducted at room temperature rather than at 100° C. It can be seen that intermediate 11 is a close analogue of the intermediate used in Montgomery's first synthesis of clofarabine. The N9 β-anomer of 11 was separated from the N9 α-anomer by a flash column and crystallized from ethanol and chloroform in almost 70% yield. The product was contaminated with a small amount of the α-anomer. Thus, it is clear the breakthrough with this procedure which leads to the much higher yield of the β-anomer being formed was a result of utilizing an SN2 reaction in the coupling step rather than SN1. Because it was difficult to remove the benzoyl groups with ammonia, sodium methoxide was used instead prior to the amination. This resulted in the halogen positioned at C6 being substituted with a methoxy group to give compound 12, in 80% yield. Amination with ammonia displaced the methoxy group to give clofarabine (7) in 78% yield. As a result of using an SN2 approach to the coupling step, the yield was improved to 47% based on carbohydrate 8, which is an overall yield of up to 18% from 8.
Although a halogen atom positioned at C6 was preferred, they also claimed alkoxy, azido, amino and protected amino groups at this position. Thus, 2-chloroadenine (13) converted to its DBU salt derivative could be coupled with 8 but this was only demonstrated on a very small scale and the yield of N9 β-anomer of 14, as isolated following preparative TLC, was a low 28%.
Despite these improvements, the main drawback of this process, being that the anomeric mixture of intermediates required column chromatography for their separation, still remained. Also the patent examples did not demonstrate that this procedure was applicable to large scale manufacture of clofarabine.

Researchers at ILEX Products, Inc. and Ash Stevens, Inc. optimized the above discussed Montgomery procedures using the potassium salt 11 of 2-chloro-6-aminopurine (a.k.a., 2-chloroadenine; 13), instead of the sodium salts of 2,6-dichloropurine (4), in the coupling with the same bromocarbohydrate 8 in a ternary solvent system in the presence of KOt-Bu and CaH2.10 The selection of 13 instead of 4 meant that Montgomery's amination step was no longer required, saving a single synthetic step. The formation of the purine potassium salt and the coupling reaction were carried out in one vessel (Scheme 8). The choice of solvent mixture had a significant influence on the anomeric selectivity and conversion. The additive CaH2 had a beneficial effect by removing trace amounts of water from the solvent. After optimization, intermediate 14 could be obtained from the coupling reaction with a 15:1 β-:α-ratio which was upgraded to an anomeric ratio of 80:1 (β/α) in 50% yield with through crystallization from butyl acetate-heptane and re-slurrying with MeOH. The deprotection of the β-enriched 14 gave crude clofarabine, which after crystallization from MeOH, gave pure clofarabine in 64% yield. The yield from carbohydrate 8 was 32%, and therefore the overall yield was up to about 14% based on starting carbohydrate 9.

Therefore, there is still need for an improved process of making clofarabine.